The present invention is directed to a method for preparing epithelial cells, particularly keratinocytes, and more particularly, primary keratinocytes, containing an adeno-assoicated virus (AAV) vector containing a gene that encodes a heterologous protein and using these transfected cells to express the heterologous protein and secrete it from the cells. The transfected epithelial cells are useful for preparing a culture of these epithelial cells that secrete the heterologous protein. The cultures are composed of epithelial cells that express the heterologous protein, and are useful in preparing sheets of epithelial cells or recombinant skin composed of stratified squamous epithelium, that express the heterologous protein. This recombinant skin is useful in skin gene therapy, as a skin graft in a subject who is in need of the expressed heterologous protein to either treat a systemic disease or a specific skin disease.
U.S. Pat. No. 4,868,116 prepared epithelial cells that expressed foreign genes by co-culturing keratinocytes and fibroblasts, also known as producer cells, that were treated to prevent their multiplication. The fibroblasts carried an infectious recombinant retrovirus containing the gene encoding the foreign gene. The keratinocytes were cultured to produce a sheet of keratinous tissue that expresses a hormone, an enzyme or a drug not normally expressed in keratinocytes. These sheets are said to be useful to graft onto subjects in need of the secreted products and to improve the general properties of the skin. However, the serious drawback of this disclosure is the use of a retrovirus as the vector to introduce the heterologous gene, which is now banned by the Food and Drug Admnistration, as a vector for gene therapy. Additionally, the co-culturing of the keratinocytes with fibroblast cells as a source of the recombinant retroviruses potentially introduces an additional cell into the skin graft that is not required in the present invention. The present invention is simpler and less dangerous as the retrovirus producer cells, the fibroblasts, are oncogenically transformed and may produce wild type (wt) retroviruses, which have been shown to produce lymphomas/leukemias. Additionally, the present invention does not select with G418 that stimulates terminal differentiation and dramatically limits the life span and growth of the keratinocytes in the skin sheets. Further, the prior art method utilizes the SV40 promoter, whereas the present invention preferably utilizes the AAV p5 and skin specific promoters, such as keratin specific promoters; for example, keratin 5 and keratin 14 promoters.
Braun-Falco et al. (20) discloses transducing human primary keratinocytes with an rAAV/LacZ construct that results in the presence of the rAAV/LacZ construct in up to 70% of human keratinocytes grown in culture. However, this study merely cultures keratinocytes, obtaining monolayers of keratinocytes, and did not produce recombinant skin that produces a heterologous protein as disclosed by the present invention. Although it is stated that rAAV shows promise as a gene transfer vehicle for skin gene therapy, there is no evidence that this system works in skin because these researchers did not produce skin. Recently several groups have found that AAV is able to infect the squamous epithelial tissues of the genital tract. (1, 4, 5, 6, 10, 16, 19).
It is an object of the present invention to produce epithelial cells containing an adeno-assoicated virus (AAV) vector containing a gene that encodes a heterologous protein.
It is an additional object of the present invention to culture the AAV transfected epithelial cells to produce a culture of epithelial cells that expresses the heterologous protein encoded by the gene.
It is a further object of the present invention to prepare recombinant skin that is an intact stratified squamous epithelium composed of these AAV transfected epithelial cells that secretes the heterologous protein for use in skin grafts to provide a subject with a source of the heterologous protein.
It is an additional object of the present invention to treat a subject in need of treatment by the expressed heterologous protein by contacting the skin of the subject with a sheet of AAV transfected epithelial cells or recombinant skin that expresses the heterologous protein into the skin of the subject.